RESUMEN
Ligation of a hairpin oligonucleotide to genomic DNA prior to bisulfite conversion and PCR amplification physically links the two complementary DNA strands. This additional step in the conversion procedure overcomes the limitations of conventional bisulfite sequencing where information of the cytosine methylation status is only obtained from one of the two strands of an individual DNA molecule. Sequences derived from hairpin bisulfite PCR products reveal the dynamics of this epigenetic memory system on both strands of individual DNA molecules. The chapter describes a reliable step-by-step procedure to generate hairpin-linked DNA. It also provides a guide for efficient bisulfite conversion that is suitable for both conventional and hairpin bisulfite sequencing approaches.
Asunto(s)
Secuencias Invertidas Repetidas/genética , Reacción en Cadena de la Polimerasa/métodos , Sulfitos/química , Citosina , ADN/genética , Metilación de ADN , ADN Complementario/química , ADN Complementario/genética , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed "hairpin-bisulfite PCR" to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of approximately 0.96 per site per cell division. For de novo methylation efficiency (E(d)), remarkably different estimates were obtained for the hypermethylated CpG island (E(d) = 0.17), compared with the hypomethylated island on the active-X chromosome (E(d) < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.